期刊
JOURNAL OF PROTEOME RESEARCH
卷 12, 期 7, 页码 3193-3206出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr400246t
关键词
cell type; astrocyte; neuron; oligodendrocyte; microglia; liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS); transcription; splicing; nucleus; neuroproteomics; epigenetic
资金
- F32 NRSA grant from the NIA [AG038259]
- NINDS [NS056097A]
- NIH [P50 (AG025688)]
- Emory Neuroscience NINDS Core Facilities P30 grant [NS055077]
The brain consists of diverse cell types including neurons, astrocytes, oligodendrocytes, and microglia. The isolation of nuclei from these distinct cell populations provides an opportunity to identify cell-type-specific nuclear proteins, histone modifications, and regulation networks that are altered with normal brain aging or neurodegenerative disease. In this study, we used a method by which intact neuronal and non-neuronal nuclei were purified from human post-mortem brain employing a modification of fluorescence activated cell sorting (FACS) termed fluorescence activated nuclei sorting (FANS). An antibody against NeuN, a neuron specific splicing factor, was used to isolate neuronal nuclei. Utilizing mass spectrometry (MS) based label-free quantitative proteomics, we identified 1755 proteins from sorted NeuN-positive and negative nuclear extracts. Approximately 20% of these proteins were significantly enriched or depleted in neuronal versus non-neuronal populations. Immunoblots of primary cultured rat neuron, astrocyte, and oligodendrocyte extracts confirmed that distinct members of the major nucleocytoplasmic structural linkage complex (LINC), nesprin-1 and nesprin-3, were differentially enriched in neurons and astrocytes, respectively. These comparative proteomic data sets also reveal a number of transcription and splicing factors that are selectively enriched in a cell-type-specific manner in human brain.
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